Relationship between the messenger RNAs transcribed from two overlapping genes of influenza virus.

The relationship of the mRNAs encoding the NS1 and NS2 polypeptides of influenza virus has been investigated through synthesis and characterisation of complementary DNA copies of the mRNAs. Previous work had shown that both mRNAs are encoded by virion RNA segment 8, and that the sequences comprising the smaller of the two mRNAs (the NS2 mRNA) were also present on the NS1 mRNA. Our results indicate that the mRNA encoding the NS2 polypeptide of the avian influenza, fowl plague virus, is approximately 400 ntds long, and that its sequences correspond largely with the 3'-terminal region of the NS1 mRNA.     

Yeast H3 and H4 histone messenger RNAs are transcribed from two non-allelic gene sets

The genes coding for the H3 and H4 histones of Saccharomyces cerevisiae have been isolated by recombinant DNA cloning. The genes were detected in a bacteriophage lambda library of the yeast genome by hybridization with plasmids containing the cloned Psammechinus miliaris sea urchin histone genes (pCH7) and the cloned Drosophila histone genes (cDM500). Two non-allelic sets of the H3 and H4 genes have been isolated. Each set consists of one H3 gene and one H4 gene arranged as a divergently transcribed pair separated by an intergene spacer DNA. The histone genes were located on the cloned yeast fragments by S1 nuclease mapping, as was a gene (SMT1) of unknown function that does not code for a histone but is closely linked to one of the histone sets. Sequence homology between the two non-allelic sets is confined to the coding regions of the respective genes while the flanking DNA and intergene spacer DNA are extensively divergent. Cellular RNA homologous to the histone genes, including transcribed non-coding sequences unique to each of the four genes, was detected by S1 mapping, thus demonstrating that all four genes are transcribed in vegetative cells.     

Intracellular vesicular stomatitis virus leader RNAs are found in nucleocapsid structures.

Previous studies demonstrated that cytoplasmic extracts of cells infected with vesicular stomatitis virus contain plus-strand leader RNAs which sediment at 18S on sucrose gradients as a complex with viral N protein. The work presented in this paper demonstrated that these 18S complexes were stable on CsCl density gradients, banding at a buoyant density near that of genome nucleocapsids, and exhibited a morphology in an electron microscope similar to the disk structures found in virus genome nucleocapsids. Minus-strand leader RNAs were also found in 18S complexes on sucrose gradients. Quantitation of intracellular leader RNA suggested that, late in infection, approximately three-quarters of total intracellular leader RNA was encapsidated.     

The genes for small nucleolar RNAs in Trypanosoma brucei are organized in clusters and are transcribed as a polycistronic RNA

Because the organization of snoRNA genes in vertebrates, plants and yeast is diverse, we investigated the organization of snoRNA genes in a distantly related organism, Trypanosoma brucei. We have characterized the second example of a snoRNA gene cluster that is tandemly repeated in the T.brucei genome. The genes encoding the box C/D snoRNAs TBR12, TBR6, TBR4 and TBR2 make up the cluster. In a genomic organization unique to trypanosomes, there are at least four clusters of these four snoRNA genes tandemly repeated in the T.brucei genome. We show for the first time that the genes encoding snoRNAs in both this cluster and the SLA cluster are transcribed in an unusual way as a polycistronic RNA.     

Oligonucleotide mapping of non-radioactive virus and messenger RNAs.

A modification of the known method for obtaining radioactive fingerprints from non-radioactive nucleic acids by labelling a digest with 5'-hydroxyl polynucleotide kinase and [gamma-32P]-ATP has been applied to RNase T1 digests from various high molecular weight virus RNAs and to ovalbumin mRNA. Fractionation of the resultant [32P]-labelled T1 RNase digests by two-dimensional polyacrylamide electrophoresis demonstrates that in the case of virus RNAs, the fingerprints thus obtained are very similar to those derived from uniformly labelled RNAs. The value of this technique is that it requires only 1-5 microgram of purified virus RNA and at least three orders of magnitude less radioactivity than is routinely employed in preparing uniformly labelled RNA.     

Axonal and presynaptic RNAs are locally transcribed in glial cells.

In the last few years, the long-standing opinion that axonal and presynaptic proteins are exclusively derived from the neuron cell body has been substantially modified by the demonstration that active systems of protein synthesis are present in axons and nerve terminals. These observations have raised the issue of the cellular origin of the involved RNAs, which has been generally attributed to the neuron soma. However, data gathered in a number of model systems indicated that axonal RNAs are synthesized in the surrounding glial cells. More recent experiments on the perfused squid giant axon have definitively proved that axoplasmic RNAs are transcribed in periaxonal glia. Their delivery to the axon occurs by a modulatory mechanism based on the release of neurotransmitters from the stimulated axon and on their binding to glial receptors. In additional experiments on squid optic lobe synaptosomes, presynaptic RNA has been also shown to be synthesized locally, presumably in nearby glia. Together with a wealth of literature data, these observations indicate that axons and nerve terminals are endowed with a local system of gene expression that supports the maintenance and plasticity of these neuronal domains.     

PIWI-interacting small RNAs: the vanguard of genome defence

PIWI-interacting RNAs (piRNAs) are a distinct class of small non-coding RNAs that form the piRNA-induced silencing complex (piRISC) in the germ line of many animal species. The piRISC protects the integrity of the genome from invasion by 'genomic parasites'--transposable elements--by silencing them. Owing to their limited expression in gonads and their sequence diversity, piRNAs have been the most mysterious class of small non-coding RNAs regulating RNA silencing. Now, much progress is being made into our understanding of their biogenesis and molecular functions, including the specific subcellular compartmentalization of the piRNA pathway in granular cytoplasmic bodies.     

Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs

Recent advances have fuelled rapid growth in our appreciation of the tremendous number, diversity and biological importance of non-coding (nc)RNAs. Because ncRNAs typically function as ribonucleoprotein (RNP) complexes and not as naked RNAs, understanding their biogenesis is crucial to comprehending their regulation and function. The small nuclear and small nucleolar RNPs are two well studied classes of ncRNPs with elaborate assembly and trafficking pathways that provide paradigms for understanding the biogenesis of other ncRNPs.     

The genome and the intracellular RNAs of avian myeloblastosis virus

Avian myeloblastosis virus (AMV) is an acute leukemia virus which causes a myeloblastic leukemia in birds and transforms myeloid hematopoietic cells in vitro. We have analyzed RNA from AMV virions and from AMV-transformed producer and nonproducer cells by gel electrophoresis followed by transfer to chemically activated paper and hybridization to several complementary DNA (cDNA) probes. Using a cDNA probe specific for AMV, we identified two RNA species of 7.2 and 2.3 kb, which were present in all AMV-transformed cells and in all AMV virion preparations examined. The 7.2 kb species, which is presumably the genome of AMV, appears to contain the entire retroviral gag gene and at least part of the pol gene, but lacks much (or all) of the env gene. Thus AMV differs from other acute leukemia viruses described to date, since the latter have genomes of 5.5 to 5.6 kb, have only part of the gag gene and lack pol sequences. The smaller RNA does not contain gag-, pol- or env-specific nucleotide sequences but does carry nucleotide sequences from both the 5' and 3' termini of the genome, suggesting that it may be a subgenomic mRNA. Both the 7.2 and 2.3 kb species were associated with the 70S RNA complex in virions. These results suggest that AMV, unlike other acute leukemia viruses, does not express its transforming gene via a gag-related "fusion" protein but rather as a (so far unidentified) protein translated from a subgenomic mRNA.     

Genes for two small cytoplasmic Ro RNAs are adjacent and appear to be single-copy in the human genome

Anti-Ro autoantibodies precipitate several small cytoplasmic ribonucleoproteins from mammalian cells. The RNA components of these particles, designated hY1-hY5 in human cells and mY1 and mY2 in mouse cells, are about 100 nucleotides long. We have analyzed a genomic clone that appears to contain true RNA-coding regions for two of the human Ro RNAs, hY1 and hY3. These RNAs exhibit many sequence and secondary structure homologies, both with each other and with the recently sequenced hY5 RNA. The hY2 RNA is a slightly truncated form of hY1; several shorter versions of hY3 are also detected in cell extracts and immunoprecipitates. The human hY1 and hY3 genes cross-hybridize with the mouse Ro RNAs, mY1 and mY2, respectively; we show that the mouse Ro RNAs are exclusively contained in Ro particles. The genes for hY1 and hY3 are transcribed in vitro by RNA polymerase III. In contrast with all other mammalian class III genes described, they appear to be present as single copies in the human genome.     

Extreme ends of the genome are conserved and rearranged in the defective interfering RNAs of semliki forest virus

We have analyzed Semliki Forest virus defective interfering RNA molecules, generated by serial undiluted passaging of the virus in baby hamster kidney cells. The 42 S RNA genome (about 13 kb ²) has been greatly deleted to generate the DI RNAs, which are heterogeneous both in size (about 2 kb) and sequence content. The DI RNAs offer a system for exploring binding sites for RNA polymerase and encapsidation signals, which must have been conserved in them since they are replicated and packaged. In order to study the structural organization of DI RNAs, and to analyze which regions from the genome have been conserved, we have determined the nucleotide sequences of (1) a 2.3 kb long DI RNA molecule, DI309, (2) 3′-terminal sequences (each about 0.3 kb) of two other DI RNAs, and (3) the nucleotide sequence of 0.4 kb at the extreme 5′ end of the 42 S RNA genome.The DI309 molecule consists of a duplicated region with flanking unique terminal sequences. A 273-nucleotide sequence is present in four copies per molecule. The extreme 5′-terminal nucleotide sequence of the 42 S RNA genome is shown to contain domains that are conserved in the two DI RNAs of known structure: DI309, and the previously sequenced DI301 (Lehtovaara et al., 1981). Here we report which terminal genome sequences are conserved in the DI RNAs, and how they have been modified, rearranged or amplified.